Alexa fluor 546 goat anti mouse antibody

For DNA synthesis studies HeLa cells growing on coverslips in a 6-well plate were treated with relevant drugs (as described in the text) and 15 minutes before fixation 10 µM EdU (Berry & Associates, PY 7562) was added. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed three times with PBS, then permeabilised by treating twice for 10 minutes with PBS-TX (PBS/0.1% Triton X-100) at room temperature. Click reaction mix (0.1 mM 6-carboxyfluorescein TEG-Azide (Berry & Associates, FF 6110), 10 mM Sodium-L-Ascorbate and 2 mM Copper-II-Sulphate) was added to the cells for 30 minutes in the dark. Coverslips were washed three times for 5 minutes with 1% BSA 0.5% Tween in PBS at room temperature in the dark to remove excess copper and azide. Nuclei were stained with DAPI and coverslips were mounted using SlowFade Gold Antifade Reagent (Invitrogen, S36936).
For pSer40/41MCM2 and γ^∼H2AX staining cells were treated and fixed as above. Primary anti-pSer40/41MCM2 and anti-γ^∼H2AX antibody (Millipore, 05-636) were used together with secondary Alexa Fluor 546 goat anti-rabbit antibody (Life Technologies, A11010) or Alexa Fluor 546 goat anti-mouse antibody (Life Technologies, A11003) respectively.

Acini were fixed with 2% paraformaldehyde to detect phospho-Histone H3 and actin, 4% paraformaldehyde to detect GM130, H2B-GFP, H2B-mCherry, and laminin, and methanol/acetone to detect E-cadherin. Anti-phospho-Histone H3 (Ser10) rabbit monoclonal antibody, goat anti-rabbit IgG Alexa Fluor 488 conjugate, and goat anti-mouse Alexa Fluor 488 conjugate were purchased from Cell Signaling Technology (Danvers, MA). Mouse anti-GM130 antibody and mouse anti-E-cadherin were purchased from BD Biosciences (San Jose, CA). Mouse anti-laminin-5 antibody Alexa Fluor 488 conjugate was purchased from EMD Millipore Corporation (Billerica, MA). Alexa Fluor 568 phalloidin, Alexa Fluor 546 goat anti-mouse antibody, and ProLong Gold anti-fade reagent with DAPI were purchased from Life Technologies (Grand Island, NY).